There is an inadequate supply of donated human soft tissues for a variety of surgical applications. Use of the patient's own tissues is often effective but is associated with morbidity or not available due to prior use or inadequate due to pre-existing disease. We have been developing new cryopreservation methods that avoid ice formation, which damages the extracellular tissue matrices, and permit low cost storage and shipping using minus 80C freezers and dry ice, respectively. Published allogeneic large animal studies have demonstrated a significant decrease in immunoreactivity and superior outcomes compared with traditionally frozen cryopreserved control tissues. Recently, we discovered that the process developed for cardiovascular tissues results in a significant decrease in the innate, cellular immune response of human peripheral blood mononuclear cells to porcine tissue as well as human tissue in vitro. These observations suggest that porcine tissues processed by our ice-free cryopreservation technology may be employed in humans. This proposal evaluates a major hurdle previously encountered in vivo for porcine tissues and organs in humans and other old world primates, the hyperacute immune response to the galactose-a(1,3)-galactose antigen (Gal) found on porcine cells. We plan performance of histological/immunohistochemistry evaluation of wild-type and control Gal knockout porcine tissues (fresh, cryopreserved by freezing, and ice-free cryopreserved) before and after subcutaneously implantation in Gal knockout recipients. Gal knockout pigs have preformed circulating antibodies to Gal, just like humans, so these genetically engineered pigs can be used to evaluate processing effects on Gal recognition by ice-free cryopreservation without recourse to primate implant models. The tissues will be explanted after 2 and 4 weeks and the cellular infiltrates of the implants will be characterized using routine stains. Immunohistochemistry will also be performed to compare the recipient's response to different processed tissue groups with emphasis on macrophage phenotype, proinflammatory phenotype (M1) versus the phenotype (M2) associated with tissue repair and constructive remodeling. If ice-free cryopreservation of wild-type porcine tissues results in a significant decrease in M1 proinflammatory macrophages and increased M2 repair response we will employ wild-type donor pigs in future phases of product research and development. If ice-free cryopreservation does not impact Gal recognitions in genetically engineered pigs with anti-Gal antibodies future product development will instead focus on use of Gal knockout tissue donors.